A gain-of-function mutation in head involution defective , Wrinkled, causes precocious cell death of wing epidermal cells in Drosophila

In Drosophila , wing epidermal cells undergo programmed cell death as the last step of metamorphosis. The aim of this study was to evaluate the role of hid , particularly the Wrinkled mutation ( hid W ), an allele of hid , in the cell death. The wing epithelial cell death is suppressed by loss-of-function mutation of hid , indicating that the death is governed by a cascade involving hid . Examination of the cell death in hid W showed that precocious death started at G stage, 3 h before eclosion. Thus, mutated-HID in the hid W mutant was activated at G stage, supporting the gain-of-function effect of hid W mutation.

. A proapoptotic gene, hid, is involved in wing epidermal cell death after eclosion A. Wing epidermal cells marked with nuclear-localized GFP in en-Gal4 UAS-GFPN flies at eclosion (a), just after wing expansion (b), and at 1 h after eclosion (c). The wing epidermal cells are present till 0 h after wing expansion, but disappear at 2 h after eclosion because of PCD. Nuclear-localized GFP is detectable only in the nuclei of vein cells (c). B. Even at 2 h after eclosion, wing epidermal cells in the hid mutant of UAS-GFPN en-Gal4/+; th hid A206 ri/mwh H99 ri were still present because PCD was suppressed. C. Wing phenotypes in a Wrinkled (hid W )/TM6b mutant at a few days after eclosion (a). The wings show wrinkled and unexpanded phenotypes. The hid W heterozygous mutant just after wing expansion (b). The wings show balloon and blistered phenotypes. After reabsorption of the body fluid, the wings shrunk to the size of those in (a). D. Wing epidermal cells in the heterozygous mutant, UAS-GFPN en-Gal4/CyO; hid W /TM6b just after wing expansion (a) and at EP (b), G (c) and S (d) stages. The Wrinkled (hid W ) mutation causes precocious cell death of wing epidermal cells after G stage. E. Transmission electron microscopy in a wing epidermal cell at 0 h after eclosion in CS (wild type) (a) and hid W /TM6b mutant (b). The arrow in (b) indicates condensed chromatin structure. Suppression of blistered wing phenotype (g) and precocious cell death (H) of hid W mutation by ectopic expression of anti-apoptotic protein p35 in en-Gal4 UAS-GFPN/+; UAS-p35/hid W . (i-k) The nuclei marked with GFPN in UAS-GFPN en-Gal4/CyO; hid W /TM6b flies were detected in allmost cells at S stage (i), but decreased in G stage (j) and EP stage (k). F. Suppression of and precocious cell death (a) and blistered wing phenotype (b) of hid W mutation by ectopic expression of anti-apoptotic protein p35 in en-Gal4 UAS-GFPN/+; UAS-p35/hid W . G. Appearance of precocious cell death at various stages of pharate adult in UAS-GFPN en-Gal4/CyO; hid W /TM6b.

Description
Programmed cell death (PCD) is important for eliminating unnecessary or deleterious cells during the building of the mature body in developmental processes (Jacobson et al., 1997, Vaux andKorsmeyer, 1999). Most physiological cell death appears to be induced via common core effectors, including caspase proteases that are central components of the machinery responsible for apoptosis. In Drosophila melanogaster, four pro-apoptotic genes, reaper (rpr) (White et al., 1994), head involution defective (hid) (Grether et al., 1995), grim (Chen et al., 1996), and sickle (skl) (Christich et al., 2002;Srinivasula et al., 2002;Wing et al., 2002a), induce cell death. The products of these cell death genes have the potential to bind to inhibitory apoptotic protein (IAP) to prevent its function (Hay et al., 1995, Vucic et al., 1997, Goyal et al., 2000. Free IAP protein can bind to and activate the initiator caspase , Meier et al., 2000. Thus, the products of these pro-apoptotic genes activate the caspase, resulting in the induction of cell death. Although these proapoptotic genes act through a similar mechanism, various signals converge onto pro-apoptotic genes via a different signaling systems in different tissues during development. How these extracellular signals and signal transduction pathways are linked with the apoptotic machinery remains unclear. After wing extension, cell death is initiated in wing epidermal cells in Drosophila (Johnson and Milner, 1987, Kimura et al., 2004, Link et al., 2007. The death of wing epidermal cells involves the breakdown of nuclear membranes, followed by the fragmentation of nuclear DNA. Transmission microscopy revealed that cell death occurs through autophagy-like processes. Ectopic expression of an anti-apoptotic gene, p35, inhibited cell death, indicating the involvement of caspases (Kimura et al., 2004). The knockout of apical caspases of the apoptosome components dark and dronc or effector caspase drice in the wing epidermal cells suppressed cell death (Xu et al., 2005, Link et al., 2007. Although death initiates after wing extension, the death competence of wing epidermis is acquired 3 h before eclosion (Kimura et al., 2004). Garcia-Hughes et al. (2015) showed that transcription of hid and accumulation of HID protein is induced prior to cell death in the wing epidermal cells, suggesting that the pro-apoptotic gene, hid, is regulated post-translationally during the wing epidermal cell death. In this study, the role of Wrinkled (hid W ), the dominant allele of hid (Abbot and Lengyel, 1991) in cell death was examined.
Wings of the newly eclosed wild-type flies are folded ( Fig. 1Aa) but expand within 30 min following a certain set of behaviors (Fig. 1Ab, Movie 1, Baker and Truman, 2002). A strain of flies carrying en-Gal4 UAS-GFPN expressed nuclear-localized GFP in epidermal cells in the posterior compartment of the wings (Fig. 1Aa,b). After wing expansion, GFP expression disappeared in the wing epidermal cells within 2 h after eclosion (Fig. 1Ac), indicating that PCD of the wing epidermal cells had occurred (Kimura et al., 2004).
To confirm the involvement of a pro-apoptotic gene, hid, in the wing epidermal cells we examined the effects of loss-offunction mutations in the pro-apoptotic gene hid . In the hid mutant of +/UAS-GFPN en-Gal4; th hid A206 ri/mwh H99 ri, many cells expressing GFP were observed even at 2 h after eclosion (Fig. 1B). This indicates that wing epidermal cell death is mediated by HID. These results are consistent with those of a previous report (Garcia-Hughes et al., 2015).
Wrinkled (hid W ), a dominant allele of hid, is a mutation resulting in an unexpanded wing phenotype ( Fig. 1Ca; Abott and Lengyel, 1991). We examined the wing extension behavior after eclosion in hid W /TM6b mutants (Movie 2). Although these mutants showed normal wing extension behavior, adhesion of two layers of the dorsal and ventral cuticles of the wings was disrupted, resulting in the formation of blister wings during extension (Fig. 1Cb). After absorption of the hemolymph, the wings became crumpled (Fig. 1Ca).
Previously, we reported that cell death in wing epidermal cells plays an important role in wing maturation, and precocious cell death results in blistered wing (Kimura et al., 2004). To examine whether precocious cell death caused blistered wing in the hid W mutant, we evaluated the fate of wing epidermal cells in en-Gal4 UAS-GFPN/CyO; hid W /TM6b flies. In the hid W mutants, cell death had proceeded even just after wing spreading (Fig. 1Da). Nuclei marked by GFP were not observed in some regions of the wings, and cells expressing GFP in the cytoplasm were scattered. Morphological studies using transmission electron microscopy revealed that the wing epidermal cell in the wild-type fly showed a healthy spherical nucleus with some electron dense materials at eclosion (Fig. 1Ea). However, some cells in the hid W mutant possessed condensed chromatin, typical dying feature, even at eclosion (Fig. 1Eb). These results indicate that hid W is a gain-of-function mutation that causes precocious cell death in wing epidermal cells.
PCD is regulated by promoting or inhibiting caspase activation in many cases (Meier et al., 2000). To determine whether caspase dominates cell death in hid W mutants, we examined the effect of forced expression of the anti-apoptotic protein baculovirus p35. p35 can act as a specific caspase inhibitor (Zhou et al., 1997). We followed the fate of wing epidermal cells in en-Gal4 UAS-GFPN/+; UAS-p35/hid W adults. During wing extension, when the cells died in hid W mutants, GFP was observed in the nuclei (Fig. 1Fa). Thus, precocious cell death by hid W was inhibited by p35, indicating that the cell death is caspase-dependent. The flies recovered from the balloon wing phenotype during wing expansion and showed a flat expanding wing (Fig. 1Fb).
In the wild-type flies of en-Gal4 UAS-GFPN, no wing epidermal cell death is seen until at wing expansion after eclosion (Kimura et al., 2004). Then, we investigated the time at which precocious cell death began in the hid W mutant (Fig. 1Db-d, G). At S stage in pharate adults (Fig. 1Dd), GFP-expressing nuclei were observed throughout the region, indicating that the cells had not died. From G stage or later, breakdown of the nuclear membrane was observed in many cells, indicating that precocious cell death was induced (Fig. 1Db, c, G). Thus, altered-HID in the hid W mutant should be activated even at G stage.
Wing epidermal cell death occurs after wing expansion. Studies using neck-ligation and hemolymph injection confirmed that a hormonal factor directly triggers cell death (Kimura et al., 2004). This hormone would be bursicon, as bursicon silencing in the central nervous system generated cell death-defective phenotypes (Garcia-Hughes et al., 2015). The hormonal signal is transmitted via the G-protein-coupled receptor of the rickets and cAMP/PKA signaling pathway (Kimura et al., 2004). After being triggered by a hormonal signal, the death of wing epidermal cells proceeds within 1 h. Rapid induction of cell death is controlled by post-translational, rather than by transcriptional regulation. Analyses of the loss-of-function of the hid mutant showed that hid is involved in wing epidermal cell death. However, wing epidermal cell death does not occur until the deathtriggering hormone is released after eclosion, even though wing epidermal cells already expressed hid mRNA and HID protein (Garcia-Hughes et al., 2015). The HID amino acid sequence contains three consensus PKA phosphorylation sites (Grether et al., 1995), suggesting that HID is a direct target of PKA. Phosphorylation of HID by PKA may activate HID.
In the hid W mutant, precocious cell death was induced at approximately G stage and later, which was similar in timing to the competence of death induced by cAMP/PKA activation (Kimura et al., 2004). Thus, the product of mutated hid W appears to be active without hormonal signals, indicating that the mutant HID W is constitutively active. Investigations on the PKA phosphorylation site in HID and the molecular nature of the gain-of-function mutation hid W would provide insight into these mechanisms.

Fly strains
Flies were raised on cornmeal-yeast medium at 25°C under constant illumination. Canton-S (CS) flies were used as the wildtype strain. To follow the fate of epidermal cells after eclosion, wing epidermal cells were marked with nuclear-localized GFP (GFPN). A strain carrying both engrailed-Gal4 (en-Gal4) (Dormand and Brand, 1998) and UAS-GFPN (Shiga et al., 1996) on the same second chromosome was used as the wild-type. The en-Gal4 line was used to express the following transgenes: UAS-p35 (anti-apoptotic baculovirus protein; Hey et al., 1995, Zhou et al., 1997 using the GAL4/UAS expression system (Brand and Perrimon 1993).
A line of Wrinkled (hid W ) mutation, a dominant allele of hid, was obtained from the Kyoto stock center. A strain carrying the loss-of-function mutation of hid; th hid A206 ri/TM6b and mwh Df (3L) H99 ri /TM6b (Abbot andLengyel, 1991, Grether et al., 1995) were a gift from H. Steller.

Observation of wing epidermal cells and detection of cell death
The wings were dissected in phosphate-buffered saline (PBS) and mounted with PBS on a slide glass. Images of expression of GFP in the posterior compartment of wing blade were obtained with the Leica (Wetzlar, Germany) confocal microscope TCS SPE using LAS AF software. The disappearance of GFPN beneath the wing hairs (each cell in wing blade produces a single wing hair) was considered to indicate cell death. Pupal stages before eclosion were classified at S, S/G, G, W, and EP under a dissection microscope in accordance to the classification provided by Kimura and Truman (1990); those stages are at about 9 hr, 6 hr, 3 hr, 50 min, and 40min before eclosion, respectively.

Histology
The wings were fixed for electron microscopy (EM) in 2.5% glutaraldehyde in 0.1 M phosphate buffer and embedded in Epon 812 using standard procedures. EM sections were stained with uranyl acetate and lead citrate and examined with a JEOL 1010 electron microscope (Tokyo, Japan).